Aim: To test the hypothesis that season affects the semen quality of swamp buffalo
(Bubalus bubalis) bulls used for artificial insemination (AI) under tropical conditions in Thailand, as it does in
Bos taurus and Bos indicus.
Methods: Clinical and andrological examinations, and monitoring of semen production and quality were carried out on five mature,
healthy swamp buffalo AI bulls in Thailand from July 2004 to the end of June 2005. Sperm output, motility, morphology
and plasma membrane integrity (PMI) were compared between three seasons of the year (rainy, i.e. July_October;
winter, i.e. November_February; and summer, i.e. March_June) with distinct ambient temperature and humidity.
Results: All bulls were diagnosed as clinically healthy and with good libido throughout the study. Ejaculate volume, pH, sperm
concentration, total sperm number and initial sperm motility did not differ between seasons, whereas PMI and the relative
proportion of morphologically normal spermatozoa were highest in summer and lowest in winter
(P < 0.05). Buffalo age, week of collection and season influenced sperm morphology
(P < 0.05_0.001). Among morphological abnormalities,
only proportions of tail defects were affected by season, being highest in the rainy season and lowest in summer
(P < 0.001). In conclusion, climatic changes did not seem to largely affect semen sperm output or viability. Although the
proportions of PMI and tail abnormalities were affected by season, they were always below what is considered
unacceptable for AI bull sires. Conclusion: Seasonal changes did not appear to cause deleterious changes in sperm quality in
swamp buffalo AI-sires in tropical Thailand.
(Asian J Androl 2007 Jan; 1:92_101)

Semen quality in bull sires reflects the degree of normality of the function of their testes, ducti epididymides and
genital tract (including the accessory sex glands). The normality of the genital system also depends on the hormonal
balance of the sire, which is sensitive to changes in health status, nutrition and management. Changes in these
conditions influence sperm output, accessory sex gland secretion and epididymal function, all of which are reflected
in the ejaculate as volume, sperm numbers or sperm
characteristics (motility, morphology, viability etc.). The
sperm quality in the collected ejaculate, regarded as the
sum of these variables, will be normative for the quality
of the processed (mostly cryopreserved) semen and,
ultimately, of the semen's fertility when used in artificial
insemination (AI). Furthermore, external cues such as
seasonality also appear to influence sexual function,
either through photoperiod [1] or through changes in
ambient temperature [2, 3]. Because spermatogenesis is
highly sensitive to even short increases in scrotal
temperature, as has been recorded in Bos
taurus AI sires kept in temperate regions [4], a significant amount of
research has been dedicated to studies on whether
semen quality in bulls is related to variations in ambient
temperature and humidity. For instance, Bos
taurus bulls have minimum sperm output during midwinter and late
summer, concomitantly with the presence of the highest
percentages of abnormal spermatozoa. Furthermore, the
ability of their spermatozoa to survive freezing is lowest
in summer [5]. The age of the bull plays a role in these
relationships; young bulls are more affected than older
ones. Species and their inherent ability to adapt to
tropical or semi-tropical environments, is another variable that
influences whether ambient temperature/humidity affects
bull reproduction. Although Bos taurus clearly suffers
from seasonal effects in a tropical environment [6], such
effects were not seen in Bos indicus under the same
conditions [6]. Nutrition levels (often recorded as body
condition), directly associated with seasonal climatic
changes under conditions of extensive rearing, have been
reported to affect semen quality in Bos
indicus in the tropics, as shown when semen samples were repeatedly
collected from the same bull over time [7].

Similar information concerning buffalo bulls is
available, but it is mostly related to the riverine,
milk-producing type, whereas data on swamp (i.e.
meat-producing) buffalo are more scarce. Most publications
reported semen characteristics of several buffalo types
held in different locations but studied single ejaculates
[8]. Studies examining the relationship between climatic
changes and semen quality have been published for
Murrah buffalo [9], Surti buffalo [10], and, at least with
regard to sperm output, also for river [11] and
swamp-type buffalo [12]. The fact that so few publications
discuss swamp-type buffaloes is not surprising because it
is difficult to obtain repeated semen samples from the
same bull, especially from on-farm, beef-producing
buffaloes. These sires are most often used for natural
mating and they are not accustomed to semen collection
by artificial vagina (AV), thus limiting the collection of
repeated samples at short intervals. An alternative way
of solving this problem is to sample from sires at AI
centres, where collection routines have been established.
However, the number of swamp buffalo sires is limited
to the size of the AI programme in place. In Thailand,
for instance, where the majority of buffaloes are of the
swamp type, there is low use of AI, mainly as a result of
the traditional husbandry of the buffaloes in the
countryside [13].

In a previous retrospective study of Thai swamp
buffalo AI bulls by Koonjaenak et al. [13], sperm quality,
defined as sperm output and motility (initial and
post-thaw), was found to vary throughout the year under the
tropical conditions of Thailand, with the sperm
concentration being highest during the rainy season and lowest
during summer. However, the data analysed included
few semen collections during some periods (1988_1993,
2001_2004 and 2004_2005) and solely included sperm
concentration and motility, but no other variables such
as clinical status, sperm morphology or viability, thus
preventing the authors from concluding that season
affects semen quality of Thai swamp buffalo.

The objective of the present study was, therefore, to
test the hypothesis that season affects swamp buffalo
semen quality in Thailand. Semen was repeatedly
collected from AI-sires available during a full year (from 1
July 2004 to 31 June 2005) and semen quality was
compared between three seasons of the year (the rainy season,
i.e. July_October; winter, i.e. November_February; and
summer, i.e. March_June), each with a distinct ambient
temperature and humidity. Apart from clinical
monitoring of the sires, we examined sperm output, motility,
morphology and the integrity of the plasma membrane
of spermatozoa.

2 Materials and methods

2.1 Location of the study

The present study was carried out at the Frozen
Semen and Artificial Insemination Centre of the
Department of Livestock Development (DLD) in Khon-Khaen
province, north-east of Thailand at latitude 16.3 N and
longitude 102.8 E.

2.2 Animals

The present study included five Thai swamp buffalo
bulls aged 10.0 ± 4.5 years (mean ± SD, range 6_18 years)
with live weight of 854.0 ± 37.0 kg (mean ±
SD, range 822.0_924.0 kg) at the beginning of the study. The
animals were fed grass(Panicum maximum
and Brachiaria ruziziensis) and commercial concentrate pellets
supplemented with minerals. The bulls were kept in sheltered
paddocks with access to a small pond and had constant
access to running water.

2.3 Clinical examination

The study started in July 2004 and was carried out
until June 2005. A clinical history of each bull was taken
at the start, including previous illnesses, mating behaviour
and libido. Body condition score (BCS) was measured
using a grading scale of 1_5, according to a current
system for bulls [14]. For the scoring, the appearance of
the tail head, brisket and hump, the transverse processes
of the lumbar vertebrae, the hips (trochanter major) and
the ribs as well as the shape of the muscle mass between
the hooks (tuber coxae) and pin (tuber ischii) were
visually assessed. On a scale of 1_5, condition score 1
indicated severe under-condition whereas score 5 indicated
severe over-condition (obesity). Scrotal circumference
(SC) was measured at the widest midscrotal point using a
standard scrotal plastic tape (Reliabull, Lene Manufacturing,
Denver, CO, USA). Testicular consistency (TC) was
determined subjectively by palpation and classified as
normal, soft or hard. BCS, SC, TC and live weight were
measured twice by the same operator, first at the
beginning of the study (July 2004) and second in January 2005
(winter season).

2.4 Semen collection and evaluation

Semen was routinely collected from all sires once a
week using an AV. For the present study, one semen
sample per bull was screened every second week.
Immediately after collection, the ejaculate was assessed by
an experienced operator for aspect (1 = clean, 2 = dirty
or contaminated), colour (1 = watery, 2 = milky, 3 =
creamy) and density (0 = thin, D = dense, DD = very
dense). Volume (mL, graduated collection tube), pH and
sperm motility were then assessed. The pH was measured using pH paper test strips (Carlo Erba, Milano, Italy),
with a range of 5.5_9.0. A light microscope equipped
with phase contrast optics was used to determine mass
activity (0 = no mass activity, 1 = slow waves, 2 = quick waves, 3 = very quick waves, × 50) and the percentage
of individual spermatozoa depicting a pattern of
progressive, rectilinear movement (× 400). Sperm
concentration was manually assessed with a
haemocyto-meter (Bürker's chamber), as described by Bane [15].
The total number of spermatozoa per ejaculate was
calculated by multiplying sperm concentration/mL by
volume (mL), and expressed as 109 total spermatozoa.

2.5 Sperm morphology

An aliquot of each ejaculate was placed into labelled
vials containing buffered formalin solution [16] and mixed
thoroughly for quicker fixation. A drop of raw semen
was placed over a labelled slide and spread
discontinuously to form dense ridges before drying (ridge smears).
Thin smears were prepared from a physiological
saline-extended semen sample of the same ejaculate and spread
out using a blunt-edged slide (thin smears). All smears
were allowed to dry and all samples taken to the
laboratory at the Faculty of Veterinary Medicine, Kasetsart
University, Nakon Prathom, for staining and sperm
morphological evaluation. The thin smears were stained with
Williams solution (carbol-fuchsin-eosin) as described by
Lagerlöf [17], while the ridge smears were stained with
hematoxylin-eosin. Sperm morphology was evaluated
on wet smears of the formalin-fixed spermatozoa and a
phase contrast microscope (× 1 000) to detect
percentages of spermatozoa with heads (including acrosome and
midpiece) and tail abnormalities as well as the presence
of proximal and distal cytoplasmic droplets on 200
spermatozoa per sample. For the evaluation of sperm head
shape morphology, a total of 500 spermatozoa per thin
slide were counted under light microscopy at × 1 000.
The presence and relative quantity of foreign cells (such
as cells of the seminiferous epithelium, epididymal cells,
epithelium of the urethra, prepuce/penis, accessory glands,
leukocytes, lymphocytes and monocytes/macrophages)
were accounted for in the ridge smears. The relative
presence of each foreign cell type was classed as 0 = absent,
1 = scarce, 2 = moderate, and 3 = rich to very rich. The
relative percentage of morphologically normal
spermatozoa was recalculated as the mean of those spermatozoa
considered to be without defects in the wet smears
(formalin-fixed) and in the Williams-stained smears.

2.6 Sperm plasma membrane integrity (PMI)

Sperm plasma membrane integrity (PMI) was evaluated using a hypo-osmotic swelling test (HOST) [18]. An
aliquot of 100 µL of semen was suspended in 1
000 µL of HOST solution (sodium citrate and fructose solution, 100
mOsmol/kg) and incubated at 35ºC for 45_60 min. After
this incubation, 300_400 µL of the sperm suspension was
fixed in a fixing medium (1 000 µL of HOST solution plus
5% formaldehyde) for later evaluation on wet smears. Two
hundred spermatozoa per smear were counted under phase
contrast light microscopy at × 400 magnification and the
percentage of typical tail coiling/swelling was determined.

2.7 Meteorological data

Ambient temperature (ºC), percentage of humidity,
and rainfall (mm) for the present study period were
obtained from Pha Phra Station of the Meteorological
Department of the Ministry of Information and
Communication Technology, Khon Kaen, Thailand. The station
was located near the bull station where the sires were
stationed. Owing to distinct mean maximum levels of
ambient temperature, rainfall and humidity, for the
purpose of the present study we arbitrarily divided the year
into three seasons, namely (i) the rainy season:
July_October; (ii) winter: November_February; and (iii)
summer: March_June (Table 1).

2.8 Statistical analysis

Meteorological data were evaluated using the general
linear model (GLM), whereas semen and sperm data were
examined using the repeated measure statement of the
MIXED procedure (Proc MIXED) of the Statistical
Analysis Systems software (SAS Institute Inc., Cary, NC,
USA). The model included the fixed effects of age of
the bull, ejaculate (week of collection), season, mean
maximum temperature, humidity and the interaction
between them. Sperm morphology, PMI data and number
of foreign cells in an ejaculate were square
root-transformed before the analysis. Pearson's correlation
coefficients were used to examine the association between
semen parameters and sire age, season and
meteorological data. A Bonferroni test was used to determine
differences between individual semen quality variables.
Differences were considered to be statistically significant at
P < 0.05.

3 Results

3.1 Clinical assessment

The BCS of the buffalo bulls at the beginning of the
study was 4 ± 0, and this score was maintained throughout
the study period. The mean weight of the bulls increased
slightly from 854.0 ± 37.0 kg at the beginning of the study
(rainy season) to 865.0 ± 42.0 kg during the winter season.
The mean SC of the bulls was 35.6 ± 1.4 cm (range
34.0_38.0 cm) and did not vary between examinations. Both TC
and elasticity were considered within normal limits and did
not differ between examinations. Despite the fact that one
of the sires (bull No. III) refused semen collection on two
isolated opportunities, libido was considered normal for
the buffalo bulls under these management conditions. All
bulls were diagnosed as healthy and free from any
clinical disorders throughout the study period.

3.2 Immediate semen analyses

A total of 118 ejaculates were collected during the
period of study. The distribution of collections per bull
and season is shown in Table 2. Three ejaculates were
considered very thin (watery) and were therefore
discarded from semen processing and freezing of AI-doses
and, therefore, from further analyses. Semen
characteristics of the remaining 115 ejaculates entering
processing, which were recorded immediately
postcollec-tion, are summarized in Table 3. Most ejaculates were
clean, dense to very dense (D = 44.1%, DD = 52.5%),
and milky (47.5%) to creamy (50.0%) in colour. The
density and colour of buffalo semen were affected by bull
age (P < 0.05), with an increase in both with age. Most
ejaculates showed mass activity, with either very quick-
(score 3: 56.8%) or quick waves (score 2: 40.7%).

As shown in Table 3, semen samples from these five
buffalo bulls had a mean ejaculate volume range of
3.2_3.8 mL, with an average pH of 6.9_7.0, across the seasons.
The average pH of semen was affected by week of
collection (ejaculate, P < 0.05). Sperm concentration ranged
from 1.1 to 1.2 billion/mL. Furthermore, the average sperm
concentration was affected by bull age (P < 0.001,
increasing with age of the sire) and week of collection
(ejaculate, P < 0.001). The average total sperm number
per ejaculate ranged from 3.6 ± 0.3 to 4.3 ± 0.3 ×
109 spermatozoa, being also affected by bull age
(P < 0.05, increasing with age) and by week of collection (ejaculate,
P < 0.05). Initial sperm motility ranged from 72.8% to
75.2%, whereas PMI ranged from 68.7% to 75.6% across
seasons. Average initial sperm motility and PMI were
affected by age (P < 0.05 and P < 0.001, respectively),
decreasing with increasing age of the bull. None of the
semen characteristics of buffalo bull showed significant
differences between seasons except for PMI, where the
mean was significantly higher in summer (P < 0.05).

3.3 Sperm morphology

Sperm morphology is summarized in Tables 4 and 5.
In the present study, the overall total mean percentages
of sperm abnormalities of buffalo bull spermatozoa were
< 15%, being (13.7 ± 0.5)% in the rainy season, (12.4 ±
0.5)% in winter and (10.7 ± 0.5)% in summer (not shown
in the Tables). The average percentage of total
pathological head shapes ranged from 2.3% to 2.4%, and of
these, spermatozoa with acrosome defects ranged from
1.1% to 1.8%. The average percentage of immature spermatozoa (e.g. with proximal cytoplasmic droplets)
ranged from 2.0% to 2.2%. Furthermore, the
percentages of total tail defects were as low as 3.2_5.3%
throughout the study period.

These results showed a total relative proportion of
normal spermatozoa ranging from 86.3% to 89.3% across
the year, the highest percentage being present in
summer (P < 0.05). The percentage of normal spermatozoa
was affected by bull age (P < 0.001, decreasing with
age), week of collection (ejaculate, P < 0.05), and the
changing seasons (mean maximum temperature and humidity,
P < 0.001).

In contrast, the mean total pathological head shapes
(%) seemed higher in the rainy season and summer than
in winter, although the amount did not differ significantly
among seasons. The average total of morphologically
deviating sperm heads were influenced by the age of
buffalo bull (P < 0.001), with the percentage being 1.5 ±
0.2% in the 6-year-old bull (Bull I), (1.0 ± 0.1)% in the
7-year-old bulls (Bull II and III), (1.3 ± 0.2)% in the
12-year-old bull (Bull IV) and (5.9 ± 0.2)% in the
18-year-old bull (Bull V). An increase was also seen with week
of collection (P < 0.05); the interaction between age and
season being significant (P < 0.05). Two
characteristics of abnormal sperm head shapes, being abnormal
contour and variable size, were found to significantly
differ among seasons. Abnormal contour was highest in
the rainy season (P < 0.05), whereas variable size of the
sperm head was highest in winter (P < 0.05).
Pear-shaped sperm heads, abnormal contour, loose abnormal
heads, undeveloped sperm heads and variable size were
affected by bull age (P < 0.001_0.05, increasing with
age), whereas ejaculate (week of collection) affected some
variables such as pear-shaped heads (P < 0.05),
abnormal contour (P < 0.001) and variable size
(P < 0.001). Furthermore, the interaction between bull age and
season affected pear-shaped heads (P < 0.05) and heads
with abnormal contour (P < 0.05).

Loose heads did not vary among seasons. However,
this variable increased with bull age (P < 0.001) and was
also affected by ejaculate (P < 0.05). Despite changing
seasons (P < 0.001), the percentage of acrosome
defects was < 2%, being only affected by bull age
(P < 0.001). The average percentage of acrosome defects appeared to be
affected by the age of buffalo bull, being (1.6 ±
0.1)% in the 6-year-old bull (Bull I), (0.2 ± 0.1)% in the 7-year-old
bulls (Bulls II and III), (0.2 ± 0.1)% in the 12-year-old
bull (Bull IV) and (1.3 ± 0.1)% in the 18-year-old bull
(Bull V).

There was no seasonal difference in the number of
immature spermatozoa, a variable affected by bull age
(P < 0.001, decreasing with age). Abnormal midpieces
did not vary between seasons but differed between
ejaculates (P < 0.05).

Tail defects in swamp buffalo AI-bull semen ranged
from 3.2% to 5.3% across seasons, being highest in the
rainy season and lowest in summer (P < 0.001).
Average total tail defect was affected by the age of buffalo
bull, being (2.3 ± 0.3)% in the 6-year-old bull (Bull I),
(3.6 ± 0.3)% in the 7-year-old bulls (Bulls II and III), (3.9 ±
0.3)% in the 12-year-old bull (Bull IV) and (7.6 ±
0.3)% in the 18-year-old bull (Bull V). The percentage of total tail
defects was affected by bull age (P < 0.001), showing
an increase with age, ejaculate (P < 0.001) and in the
interaction between age and season (P < 0.05). Among
the tail defects, the percentages of spermatozoa with
simple bent tails and coiled tails under the head were
lowest in summer (P < 0.001_P < 0.05) and were found
to be affected by bull age (P < 0.001).

3.4 Number of foreign cells in the ejaculate

Throughout the study period, the ejaculates
consistently had three types of foreign cells; epithelial,
boat-shaped and spermatogenic cells (Table 6). The very low
proportion detected was noticable (< 1%). Epithelial and
boat-shaped cells were found in all buffalo bull ejaculates,
whereas spermatogenic cells were found only in the
semen of bulls No. IV and V. Neither epithelial nor
spermatogenic cells differed significantly between seasons,
but presence of boat-shaped cells was lowest in the rainy
season (P < 0.05).

4 Discussion

In the present study, we examined the production of
semen in swamp buffalo sires for freezing-thawing and
ulterior use for AI in Thailand over a complete 12-month
period. The sires were healthy during the whole study
period, providing ejaculates with similar pH values
(6.9_7.0) across the seasons [11, 19]. Ejaculates had an
average volume of 3.0_4.0 mL, containing 3.5_4.5 billion
spermatozoa with good viability and motility (> 65% and
> 70%, respectively). Furthermore, the total percentage
of morphologically abnormal spermatozoa was < 15%, a
figure considered normal for AI-sires of the bovine
species. The data suggest that sperm quality in swamp
buffalo AI sires, herein defined as sperm concentration,
total spermatozoa per ejaculate, initial sperm motility and
overall sperm morphology, did not vary statistically across
the year under tropical conditions in Thailand. Some
individual sperm defects such as the proportions of sperm
tail abnormalities, as well as the proportions of
spermatozoa with intact membranes, showed significant
variations over the year, however, with bull age and week of
collection being the factors influencing these variations.

Ejaculate volume has been reported to increase with
age in Malaysian swamp buffalo [8, 20]. The average
semen volume registered in the present study was higher
than previously reported in younger swamp buffaloes in
Thailand [12], and similar to that reported for bulls of
similar age in both swamp [8, 20] and riverine buffaloes.
In the latter category, studies have been conducted in
Murrah [21], Nali-Ravi [22] and Surti breeds [10].
Ejaculate volume has been reported as not being
influenced by seasonality in buffaloes generally [8] and in
Murrah [9] or Nali-Ravi buffalo breeds specifically [22],
or to show inconsistent variations that are highest in
summer [11]. These differences might be related to the age
of the buffalo bulls, differences between species,
number of specimens, management and environment
conditions during each study period.

Sperm concentration per mL (1.0_1.2 billion/mL) was
within expected limits [8, 12]. Although it seemed higher
in the rainy season and winter, our results showed no
significant seasonal differences, thus deviating from other
findings in the literature [11, 12] including our previous
results [13]. Such maintenance in sperm concentration
across seasons in the present study indicates that
seasonal changes did not affect testicular production during
the year. The differences between this and other studies
might be the result of a lower number of observations
and the length of the study period, as well as differences
in the age and breed of the sires.

Total sperm number per ejaculate obviously followed
the same trend as sperm concentration, because neither
sperm concentration nor volume differed significantly
among seasons. Total sperm number per ejaculate clearly
differed from that in other studies in the literature, where
both other variables also differed [12].

The average percentage of initial progressive motile
spermatozoa during the present study period surpassed
70%, a figure considered normal for swamp buffalo [8,
12, 20] and Murrah buffalo [9]. Despite slight
differences between seasons, these were not significant,
confirming previous results in Murrah [9, 11] and Surti
buffalo [10]. Differences between seasons have been
reported, but with confounding results, either to be highest
(P < 0.05) in winter compared with summer [12] or to
be lowest in autumn (P < 0.05) [9]. Because sperm
motility was subjectively determined by microscopic
examination of a drop of fresh semen, these data should
be considered with caution. Nevertheless, considering
the low number of abnormal spermatozoa present in the
ejaculates of the sires in the present study, the motility
results appear convincing.

A HOST was used to assess PMI and, indirectly, to study
sperm viability (i.e. presence of live spermatozoa). The
average PMI ranged from (68.7 ± 2.0)% to (75.6 ±
2.1)%, figures close to earlier observations using eosin_nigrosin in
swamp and riverine buffalo [11, 20], studies in which
differences were seen among seasons. In the present study,
PMI was highest in summer (P < 0.05), as was the total
relative proportion of normal spermatozoa (P
< 0.05). Regarding the latter, our results differ from the literature,
where the average number of live spermatozoa was
lowest in summer (P < 0.05) [1, 11, 12]. Such differences
could have been the result of sheltering and best possible
management of the present sires, which were not
negatively affected by higher temperatures or humidity.
Furthermore, the present study found a slightly negative
significant relationship between PMI and mean maximum
relative humidity in summer (r = _0.40,
P < 0.05).

Initial sperm motility was consistently higher than
PMI during the rainy season and winter. Such
difference between motile and membrane-intact cells is not
new [20] but it is usually reversed because some spermatozoa, despite being alive, are immotile at certain
moments. The methods used for the screening of
motility and PMI are basically different in their degree of
subjectivity; sperm motility being recorded on living cells
and PMI being registered on fixed cells, the latter
providing an expected better degree of "objectivity". The
drawback for the PMI assessment is, however, that the
number of spermatozoa assayed in the HOS test used is
usually low. A slightly positive, significant relationship
was found between sperm motility and PMI (r =
0.30, P < 0.05). An objective assessment of sperm motility
using a computer-assisted semen analysis (CASA)
instrument and of PMI using flow cytometry of
fluorophore-loaded spermatozoa should provide more accurate and
detailed results. However, these instruments are costly
and not readily available at the site of collection of
buffalo semen.

The mean total relative proportion of
morphologically normal spermatozoa was high (86.3_89.3%), and
highest in summer (P < 0.05). The overall mean
percentage of abnormal spermatozoa was consistently low,
well below what is considered normal for dairy bulls [23]
and without significant differences among seasons. These
results are consistent with those found in the literature
[8, 12] reporting a healthy buffalo bull to have between
10% and 15% of total sperm abnormalities in his ejaculate.

Among abnormalities, tail defects appeared to vary
significantly among seasons, being highest in the rainy
season and lowest in summer (P < 0.001). Such
variation has not been registered previously [12] and we have
no explanation for this finding except that comparisons
must consider type of buffalo, age and environmental
conditions during each study period. The abnormalities
of sperm head and tail were affected by age
(P < 0.001), with an increase with higher age. Gupta
et al. [10], Pant [24] and Wenkoff [25] all reported that ageing in bulls
might lead to a higher incidence of morphological
abnormalities in semen. In the present study, such a
relationship was present among the buffalo sires.

In conclusion, the changing seasons in Thailand
during the period of study did not seem to affect sperm
production or the overall quality of the spermatozoa in
swamp AI buffalo sires, indicating that they tolerated the
changes in environmental temperature and relative
humidity well. However, the methods used in the present
study do not necessarily imply that changes could be
seen when the spermatozoa are stressed by extension,
cooling, freezing (cryopreservation) and thawing for AI;
procedures that followed after the examination of the
ejaculates hereby used. Therefore, more refined
methods need to be used to determine changes in sperm quality,
such as CASA and assessment of membrane integrity and stability with fluorophores, and of the sperm
chromatin resistance to controlled DNA-denaturation
challenges in cryopreserved buffalo semen.

Acknowledgment

The authors thank Mr Ayuth Harintharanon, Mrs Rapiphan Uavechanichkul and the Bureau of
Biotechno-logy for Animal Production, Department of Livestock
Development, Bangkok, Thailand, for providing
information and semen samples. Appreciation is also
expressed towards the staff members at Khon-Kaen AI
Station for help during the collection of semen samples.
Thanks also go to the Centre of Agricultural
Biotechno-logy and Faculty of Veterinary Medicine at Kasetsart
University for support in this study. This study received
financial support from the Asia-Link Project titled
"Reproduction biotechnology: modern technology to improve
livestock production under traditional Asian conditions"
and from the Swedish University of Agricultural Sciences
(SLU) in Uppsala, Sweden.